Austria says it has detected the potentially lethal strain of bird flu in several live cats.
More than 150 cats have been living at the shelter
The discovery, in the southern state of Styria, follows the detection of H5N1 in a dead cat in Germany last week.
That case was thought to be the first example outside Asia of the virus crossing species to infect a mammal.
However, it is unlikely the virus is spreading between cats and chances are negligible they may pass it to humans, says the BBC's Ania Lichtarowicz.
The virus has also spread for the first time to Poland, while Serbia says it has detected the strain in dead swans and has sent samples for further tests.
The H5N1 virus can be caught by humans who handle infected birds, but it is not yet known to have passed from one person to another.
However, experts fear the virus could mutate to gain this ability, and in its new form trigger a flu pandemic that could kill millions.
Austrian officials said the three cats were from an animal shelter near Graz that took in H5N1-infected birds last month, when the country's first case of the strain in birds was detected.
All the cats at the Noah's Ark shelter have been put in quarantine and the shelter has been closed until further notice.
Officials in Serbia say they have detected H5N1 in swans found dead in northern and western parts of the country, although samples have been sent to the UK for confirmation.
Poland's national laboratory said it had identified the virus in birds found near a river in the northern city of Torun.
Jan Zmudzinski, deputy director of the Pulawy laboratory, said that the virus was "definitely H5N1".
Samples would now be sent to the EU laboratory in Weybridge, UK, for further examination, he said.
Authorities in the country had been on the lookout for bird flu after outbreaks of H5N1 were reported across Europe in recent weeks.
Concerns had risen since Friday when a dead swan with the H5N1 virus was found on the River Oder, the border between Poland and Germany.
Germany confirmed its first cases of the virus in February.